The number of solar UV-induced skin cancers is greater than all other types of cancers combined. A better understanding of the underlying biology is needed to develop novel approaches to address this problem. UV irradiation instantaneously generates two structurally distinct types of DNA lesion: cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP). Replicating cells rapidly respond to these deleterious lesions by activating ATR kinase and its downstream target Chk1. However, it is not clear whether these lesions have different effects on DNA replication and/or ATR-Chk1 activation. To address these questions, in Aim 1, we will generate cells that have one, both, or neither type of DNA lesion by combining lesion-specific photolyases with multi-parameter flow cytometry. The proposed studies will test the hypothesis, supported by preliminary data, that there are striking differences in the mechanisms and impact of these two lesion types on DNA replication and UV-DNA damage responses. Human epidemiological studies and mouse in vivo data demonstrate that intake of caffeine (a non- selective ATR inhibitor) prevents UV-associated skin cancers. Indeed, we estimate that 260,000 skin cancers are prevented annually in the U.S. by caffeinated beverage intake. This cancer-preventive effect of caffeine could be further optimized by an improved understanding of its mechanism including whether ATR should be inhibited immediately after UV or long after UV. In Aim 2, we will determine the mechanism by which ATR inhibition suppresses UV carcinogenesis. We will test the hypothesis that immediate (not delayed) ATR inhibition reduces the mutation burden in skin by blocking error-prone lesion bypass. ATR pathway activation requires recruiting more than 10 proteins to a site of DNA damage. Theoretically, it is possible to target any of these proteins to inhibit the ATR pathway, with likely differences in the ability to sensitize to DNA damage. However, current approaches to block this pathway are limited to ATP-competitive inhibitors that target the kinase activity of ATR or Chk1. To identify other druggable mechanisms in this pathway, we performed a cell-based high-throughput screen and identified several small molecules that inhibit the ATR pathway through mechanisms other than ATP competition. One compound (ARPIN) displayed an important mechanism not shared by any other inhibitor: blockage of Chk1 release from chromatin following damage. In Aim 3, we will use this unique biological effect of ARPIN as a tool to dissect the fundamental mechanism by which Chk1 is normally released from chromatin in order to carry out its effector functions. The three proposed Aims will markedly advance our fundamental understanding of how the replication checkpoint functions, characterize a mechanistically novel inhibitor of the pathway, and provide insight that can be directly translated to further augment an already extensive public health impact. The studies will be highly collaborative with five scientists whose expertise will facilitate critical innovative aspects of te proposed Aims.